Mineral-protein preparations (MPP) and neuropathies in diabetes

ABSTRACT

The increased level of glucose in blood is the result of the destruction of β cells of the Islets of Langerhans by autoimmune process in insulin-dependent form of diabetes or a relative lack of insulin in insulin-dependent form of diabetes. Constantly present hyperglycemia in diabetes and a relative lack of insulin aid the development of the neuropathy as a late complication of diabetes. For that purpose, a technical problem was set before the inventor with the request of the reduction of glucose in blood, and stopping of the process of the development of the neuropathy as the consequence of the “long” duration of diabetes. Such a mineral-protein preparation was prepared by which glycemia was successfully regulated, but the process was also stopped by depositing of Ca +  in nerve cells. Those early changes caused by diabetes were successfully stopped, so that the appearance of the neuropathy, as a late complication in diabetes, was postponed. The mineral-protein preparation showed a hypoglycemia effect and stopped the development and progressing of the diabetic neuropathy.

FIELD TO WHICH INVENTION REFERS

This invention refers to the preparation consisting from syntheticzeolites and selected herbal proteins (MPP) which is applied forprevention of the development of neuropathies in persons suffering fromdiabetes. The present MPP stops the process of apoptosis caused byaccumulation of Ca²⁺ ions in cytoplasm of nerve cells.

INVENTION

The technical which was set before the inventor, and the solution ofwhich is presented in this patent application, consists from the MPPwhich is applied for prevention of the development of neuropathies inpatients suffering from diabetes, which will have the followingcharacteristics that:

-   1) it contains plant proteins,-   2) it reduces the concentration of glucose,-   3) it can stop the process of depositing of Ca²⁺ ions into nerve    cells,-   4) it stops the process of apoptosis in nerve cells stimulated by    depositing of Ca²⁺ ions,-   5) the process of phagocytosis of apoptotic bodies is absent,-   6) it stops the process of creating immunoglobulin on myelin fibres    of the nerve membrane,-   7) it reduces the depositing of immunoglobulin on nerve fibres,-   8) it reduces the immunological destruction of nerve fibres mediated    by the complement,-   9) the pain caused by diabetic neuropathy is absent,-   10) it contains substances for elimination of heart troubles,-   11) it contains substances for elimination of digestive troubles,-   12) it contains substances for elimination of free radicals,-   13) it contains the daily dose of B complex vitamins,-   14) it has a pronounced capability of ionic exchange,-   15) it does not pass through villi,-   16) it has a pronounced ionic exchange through the intestine-blood    barrier,-   17) its particles are of micron and submicron size,-   18) it is chemically treated,-   19) it reduces the concentration of Na⁺ ions,-   20) it is enriched with Ca²⁺ ions,-   21) the dose is reduced in relation to the natural zeolite,-   22) it is suitable for oral administration,-   23) no noticeable harmful side effects appear even with large daily    doses, and in case of a long-term use,-   24) it is not toxic,

INVENTION BACKGROUND

Diabetes mellitus is a syndrome of disturbed metabolism of glucose whicharises due to hyperglycemia which is responsible for the majority ofsymptoms. Insulin is actually the key regulator of the metabolism ofhydrocarbons, proteins and lipids, and a relative or absolute shortageof insulin effects the complete intermedial metabolism. It is supposedthat biochemical disturbances are primarily determined by the amount ofthe insulin deficiency.

Diabetes of Type I, i.e. IDDM (Insulin Dependent Diabetes Mellitus) ischaracterized by progressive autoimmune process of the destruction of βcells of Islets of Langerhans by T lymphocytes (Eisenbarth, G S. NewEngl. J. Med. 314:1360–1368, 1986). More precisely, IDDM is the resultof the destruction of beta cells mediated by CD4+ and CD8+ cells and thefunction of antigen presenting cells (APC) (Frque F., Had{hacek over(z)}ija M., et al., Proc. Natl. Acad. Sci., USA, 91:3936–3940, 1994).

NOD (non-obese diabetic) mice develop the classical picture of diabeteswhich is completely identical to IDDM in people (Makino S., et al.,Expl. Anim. 29: 1 (1980)). Furthermore, chemical diabetes caused byAlloxan in mice also develops the picture of diabetes with all theaccompanying symptoms identical to the human form of diabetes of Type I(Dunn J. S., et al., Lancet II: 384–387, 1943).

The purpose of the therapy of diabetes is the normalization of thefollowing parameters; the concentration of glucose in blood, theconcentration of lipids, and the absence of glucose and acetone inurine.

In the therapy of diabetes, two principles of treatment are applied:firstly, the general one, and secondly, special principles of treatmentof diabetes.

General Principles of Treating Diabetes

By treating diabetes, it is endeavored to achieve the state ofnormoglycemia and thus prevent the development of later complications.The general principles of treatment comprise, regardless of the type ofdiabetes: diet, physical activity, education and self-control. Thenourishment must be composed so that it meets the daily needs fornutrient according to the age, sex, activities, height and weight. It isrecommended that the diet consists of: 15–20% of proteins, 25–30% offats, and 55–605 of hydrocarbons. The total daily quantity of substancesmust be divided into a larger number of smaller meals (5–6 during theday) in order that no major oscillations in the concentration of glucosein the blood would occur, the physical activity is important because itincreases the exploitability of glucose in peripheral tissues with aproportionally reduced use of insulin. Education and self-control areimportant for understanding of the very disturbance, and for the dailyself-control of the concentration of glucose in the blood.

Special Principles of Treating Diabetes

Treatment by exogenous insulin is applied in case of Type I Diabetes,where over 805 of the mass of the endocrinous part of the pancreas. Thesuccessfulness is high since, before the discovery of insulin, suchpersons used to die after 1.5 years, and today they live for even 50years after the appearance of the disease.

Oral antidiabetics are not a replacement for insulin, but a support tothe secretion of endogenous insulin or its hypoglycemic effect. Personswith the Type II Diabetes should take oral antidiabetics only in case ifthe correct nourishment and the corresponding physical activity have notgiven the desired results. Although the transplantation of pancreas,i.e. Islets of Langerhans, is one of the special principles of treatmentof Type I Diabetes, it is still being researched today. Namely, thesuccess of the transplantation depends on the number of transplantedislets, by which the isolation is still a problem, as well as theimmunological reaction of rejection due to which variousimmunosuppressive must be used on a daily basis.

Metabolic Effects of Insulin

Insulin is a protein, with the molecular mass of 5734 Da. It consists of“A” and “B” chain, which are connected by two disulfide bridges, whilethe third disulfide bridge is within the “A” chain. “A” chain consistsof 21 amino acids, and the “B” chain of 30 amino acids.

Insulin is synthesized by β cells of the Islets of Langerhans of thepancreas. The basic physiological function of insulin consists inmaintaining of normoglycemia. Thus, insulin effects the metabolism ofhydrocarbons so that it firstly suppresses the creation of glucose inthe liver, and that happens if the concentration of insulin in thecirculation ≈30 mU/L, and secondly so that it stimulates entering ofglucose into peripheral tissues (by speeding up the translocation of theglucose transporter GLUT-4 on the surface of cells of skeletal musclesand of adipose tissue, and by activation of intracellular enzymes suchas, e.g. glycogen synthetase). That happens if the concentration ofinsulin in the circulation ≈100 mU/L, which is usually present in theblood after a meal. Insulin is also a very potent inhibitor oflipolysis, and thereby also of the ketogenesis. The antilipolitic effectof insulin is manifested already at the insulin concentration of ≈10mU/L. Insulin also inhibits the proteolysis, if its concentration in theblood is between 10–30 mU/L.

By binding of insulin to the extracellular α-subunit of receptor, theinsulin-receptor complex activates the Zn²⁺ dependent protein tyrosinekinase which makes the transmembrane β-subunit of insulin receptor and,due to that, autophosphorylation of receptor and other proteins withphosphate groups over ATP (Reddy and Kahn, 1988) arises. The activationof phosphatidylinositol-specific phospholipase C leads towards thehydrolysis of membrane phosphoinositides. Thus, the cyclicinositol-phosphate glucosamine arises, the second messenger whichactivates phosphodiesterases, reduces the content of the intracellularcAMP and produces diacylgycerol which activates protein kinase C(Saltiel, 1986). Protein kinase C regulates numerous enzymes and thevery insulin receptor through phosphorylation (Van de Werve, 1985).

During testing of Type I Diabetes by insulin, the slow absorption ofinsulin from the subcutaneous tissue results in its inadequate pique atthe time of the meal and after taking it with hyperglycemia between twomeals (due to a low concentration of insulin in the port vein). Insulindeficiency leads to an increased release of glucose by the liver, andthat is the reason of hyperglycemia on an empty stomach, i.e. ofpostprandial hyperglycemia. The consequence of the low concentration ofinsulin includes the increase of the secretions of glucagon from α-cellsof the Islets of Langerhans of the pancreas.

Insulin resistance is often joined with the appearance of obesity, theintolerance of glucose, hypertension, dyslipidemy, disturbances in bloodcoagulation and the speeded up aterogenesis and it is also referred toas the Syndrome X.

Role of Plant Proteins in Treatment of Diabetes

The results of sequencing of the human genome just published stimulateresearches about the role of small target molecules and proteinsconnected with diseases (Engl. target).

The completed sequencing of the human genome revealed a number of“harmful” genes on all chromosomes. Those results indicate and reveal anumber of target places for medicines. In that context, a new strategyof research for obtaining of new medicines is started. That strategyfirst comprises target places (primarily, those are proteins, but thatalso includes parts of nucleic acids), and secondly, the ligand, i.e.proteins of small molecules as a substitute to the “ill” target place.

Plants contain four main types of molecules: hydrocarbons, proteins,nucleic acids and lipids. Beside the stated ones, plants contain othertypes of molecules in smaller quantities, e.g.: alkaloids, terpenoids,phenols, sterols another so-called “secondary metabolites”.

Hydrocarbons in plants include monomers called monosaccharides andpolymers called polysaccharides. Polysaccharides which are included inthe construction of the plant cells are called structuralpolysaccharides. The best known structural polysaccharide in plants iscellulose (it makes 40%–60% of the cell wall). Reserve polysaccharidesserve as the reserve of food, and the best known in plants are starchand insulin.

After cellulose, proteins make the largest remaining part of the biomassof the living plant cell. Proteins in plant cells are made of 20 variousamino acids bound into polypeptides. As well as in case of hydrocarbons,proteins also have an important role in the construction of the cell(structural proteins), and they also serve as a reserve. Unlikehydrocarbons, proteins can also be enzymes. Structural proteins of thecell wall are called extensions and have an important role in itsexpansion during the very growth of the plant. Extensions are proteinsrich in hydroxyproline, serin, threonine and the asparaginic acid. Plantcells contain various kinds of membranes, each has a different proteinsystem. E.g. the inner membrane of mitochondrions and chloroplastscontains about 75% of proteins while the membrane which surrounds theplant cell has about 50% of proteins. The majority of the non-proteinpart of the membrane which surrounds the plant cell is made by lipids.The reserve proteins in plants are most often present in seeds and theyserve as the source of nutrients during germination. The contents ofproteins in seeds depends on the plant species. Some reserve proteins ofplants are: zein, gliadin, ricin D, abrin, etc. Many plant proteins havethe function of enzymes and catalyze biochemical reactions. One exampleis α-amylase, the enzyme which disintegrates amylose.

Plant Peptides and Amino Acids

In 1981, Khanna et al. published the results of the research aboutisolation of “polypeptides p” from the fruit and seeds of the plantMoomordica charantia L. (Cucurbitaceae). “Polypetide p” consists of 17various amino acids and has the molecular mass of 11,000. When the s.c.is applied in rodents, primates and people, its insulinomymetic effectin the dose of 0.5 units/kg (1.8 mg/ml=40 units) is noticed. Sulfoxideamino acids: s-methylcysteine sulfoxide (SMCS) and S-allylcysteinsulfoxide (SACS) isolated from plants Allium cepa L. and Allium sativumL. have caused the loss of body mass and the content of glycogen inliver after a month of per oral application in rats with experimentallyinduced diabetes (Sheela et al., 1995).

In 1998, Sauvaire et al. have isolated, and in 1999, Broca et al. havedescribed the in vivo effect of 4-hydroxyisoleucine as a new stimulatorof the insulin secretion from the seeds of Trigonella foenum graccum L,4-hydroxyisoleucine aids the glucose induced secretion of insulin bothon the model of isolated Islets of Langerhans, and in people. Itsstimulating effect in a dose of 100 μmol/L to 1 mol/L was dependentexclusively on the stimulation of the secretion of insulin by glucose,i.e. it did not show the activity in case of low concentrations ofglucose (3 mmol/L) or the basal concentration of glucose (5 mmol/L).

The fruit of the plant Blighia sapida Koenig (Sapindaceae) containsemetic ingredients: hypoglycine A and its γ-L-glutamyl dipeptide,hypoglycine B, which indicate a hypoglycemic activity, they act in sucha way that they inhibit oxidation of long-chain fatty acids. HypoglycineA is twice stronger hypoglycemic compound than hypoglycine B which isalso a teratogen, and thus too toxic for therapeutic use (Tanaka et al.,1972; Oliver-Bever and Zahnd 1979).

Development of Neuropathy in Diabetes

Neuropathy is a common late complication of diabetes which affectssomatic and autonomic peripheral nerves. Neuropathy occurs in a certainpercentage in Type I and Type II Diabetes (Greene, D A, et al., DiabetesCare. 15:1902–6, 1992). Peripheral nerve abnormalities in people and inthe animal model of diabetes are manifested as the decreasedconductibility of nerves, axonal reduction, and nerve fiber loss (BehseF F, et al., J. Neurol. Neurosurg. Psych. 40:1072–82, 1977; Brismar. T.Metab. Clin. Exp. 32:112117, 1983; Sima, A A F, et al., Ann. Neurol.18:21–29, 1985), in connection with metabolic alterations (Green, D A,et al., Diabetes 37:688–693, 1988), including altered calcium signaling(Levy, J, et al., Am. J. Med. 96:260–273, 1994). Existing studiesindicate that altered homeostasis of calcium ions is a widespreadoccurrence in IDDM and NIDDM. Both, in people suffering from diabetes,and in animal models of diabetes, the identical change was observed,which is the increase of the Ca2+ ions in the cytosol (Hall, K E, etal., J. Physiol. 486:313–322, 1995; Nobe, S, et al., Cardiovas. Res.24:381, 1994.; White, R E, J. Pharmacol. Exp. Ther. 253:1057–1062,1993.; Kappelle, A C, et al., Br. J. Pharmacol 111:887–893, 1992). Theincrease of the concentration of calcium ions aids the process ofnatural atrophy (apoptosis) of nerve cells, but that process has beenshown in a number of other experimental models as well (Trump, B F, etal., FASEB J. 9:219–228, 1995; Down, D., Phosphoprotein Res. 30:255–280,1995; Joseph, R, et al., Mol. Brain. Res. 17:70–76,1993).

The latest researches suggest that “serum factors” have an importantrole in the pathogensis of diabetic neuropathy in patients with Type Idiabetes mellitus. By incubation of β-cells of the Islets of Langerhansin conditions of the general tissue culture, when the serum of patientswith Type I or Type II Diabetes was added to the medium (Had{hacek over(z)}ija, M, et al. Period. Bio 1.97:313–317, 1995), apoptosis in β-cellsof the Islets of Langerhans is connected with the increase of theconcentration of L-type calcium ions (Juntti-Berggren, L, Science261:86–89, 1993). It was also shown that neuroblastoma cellsdemonstrated a reduced growth, the increase of entering of Ca²⁺ ions,i.e. the intensified apoptosis if they were exposed to the serum ofpatients suffering from Type I Diabetes with neuropathies (Pittinger, GL, et al., Diabetic. Med. 10:925–932, 1993; Pittinger, G L, et al.,Diabetic Med. 12:380–386, 1995: Pittinger, G L, et al., J. Neuroimmunol.76:153–160, 1997; Migdalis, I N, et al., Diabetes. Res. Clin. Pract.49:113–118, 2000). The complement-independent, Ca²⁺-dependent inductionsof apoptosis of nerve cells aid the appearance of autoimmuneimmunoglobulins in diabetes on nerve fibres (Srinivasan, S M, J. Clin.Invest. 102:1454–1468, 1998).

the characteristics of zeolites, such as the possibility of ionicexchange, existence of in intercrystalline pores which let throughmolecules of various dimensions, the existence of strong acidic placesand places active for the reactions catalyzed by metals, etc. make themvery interesting for a wide industrial implementation, as well as forfundamental researches (Flanigen, E. M. in: Proc. Fifth. Int. Conf.Zeolites (Ed. L. V. C. Rees), Heydon, London-Philadelphia-Rheine, 1980,p. 760; Vaughan D. E. W., Chem. Eng. Prog., 25, 1988; Cornier, J., Popa,J. M., Gubelman, M, L'actualite, 405, 1992; Subotić, B., Bronić, J.,{hacek over (C)}i{hacek over (z)}mek, A., Antonić, T., Kosanović, C.,Kem. Ind. 43:475, 1994). The interest for using zeolites as catalysts,absorbents and means for softening of water in detergents has largelyincreased in the last three decades (Cornier, J., Popa, J. M., Gubelman,M., L'actualite, 405). There are annually millions of tons of zeolitesused in the production of washing means (Cornier, J., Popa, J. M.,Gubelman, M., L'actualite, 405), hundreds of thousands of tons in oilprocessing and in the petrochemical industry (Naber, J. E., de Jong, K.P., Stork, W. H. J., Kuipers, H. P. C. E, Post, M. F. M., Stud. Surf.Sci. Catal., 84C:2197, 1994), and the use in other fields is increasingtoo (Flanigen, E. N. in: Proc. Fifth. Int. Conf. Zeolites (Ed. L. V. C.Rees), Heyden, London-Philadelphia-Rheine, 1980, p. 760; Waughan, D. E.W., Chem. Eng. Prog., 25, 1988; Cornier, J., Popa, J. M., Gubelman, M.,L'actualite, 405, 1992; Naber, J. E., de Jong, K. P., Stork, W. H. J.,Kuipers, H. P. C. E, Post, M. F. M., Stud. Surf. Sci. Catal., 84C:2197,1994.; Breck, D. W. in: The properties and Application of Zeolites,Special Publication (Ed. R. P. Towsand), 33:391, 1980.; Vaughan, D. E.W. in The properties and Application of Zeolites, Special Publication(Ed. R. P. Towsand), 33:294, 1980.; Flanigen, E. M., Pure Appl. Chem.,52:2191, 1980.), including the use of zeolites in medicine, agricultureand cattle breeding (Ramos, A. J., Hernandez, E., Animal Feed Sci.Technol., 65:197, 1997; Eriksson, H., Biotechnology Techniques 12:329,1998.; Mumpton, F. A., J. Nat. Acad. Sci., 96:3463, 1999.).

Zeolites or molecular sieves are hydrated natural and syntheticaluminosilicate compounds with a unique spatial-network structureconsisting of SiO₄ and AlO₄ tetrahedrons linked through common oxygenatoms (Breck, D. W., J. Chem. Educ. 41:678, 1964), as it isschematically presented in FIG. 1.

The negative charge of the aluminosilicate structure is caused by anisomorphic replacement of the silicium with the valence of four byaluminum with the valence of three is neutralized by hydrated cation(Na⁺, K⁺, Ca²⁺, Mg²⁺ etc.). In reality, SiO₄ and AlO₄ do not createsingle-dimensional chain structures in the structure of zeolites, as itis simplified in FIG. 1, but they create two-dimensional andthree-dimensional basic structural units, the combination of which givesrise to three-dimensional spatial-network structures characteristic forzeolites (FIGS. 2–5). The specific structure of zeolite, unique, both inthe relation with other aluminosilicates, and in relation to othercrystal materials, is reflected in existence of structural cavitiesmutually linked by channels of a certain form and size (FIGS. 2–5).However, unlike other porous materials characterized by a specifiedarrangement of pores which are statistically distributed in variousdirections, the form and size of cavities and channels, as well as theirmutual relationships are constant and exactly defined as structuralparameters of a specific type of zeolites (Barrer, R. M., Zeolites andClay Minerals as Sorbents and Molecular Sieves, Academic Press, London,1978, p. 23), as can be seen in the stated examples of the structure ofa unit lattice of the zeolite A (FIG. 2), faujasites (X and Y zeolites,FIG. 3), mordenite (FIG. 4) and the ZSM-5 zeolite and silicalite (FIG.5).

The chemical composition of zeolites is usually presented by a generalformula in the oxide form:M_(2/n).Al₂O₃ *ySiO₂-zH₂OWhere n is the charge number of cations M, while y≧2 and z depend on thetype of the zeolite. The “zeolite” water results from hydrationmembranes of the compensation M cations. Therefore, the value z dependson the type of the M compensation cations, the number of M cations in aunit zeolite lattice and the level of M cations hydration in the zeolitelattice. By heating of the zeolite up to approx. 600° C., the “zeolite”water can be removed from the zeolite without the change of thestructure. By cooling to the room temperature, the same quantity ofwater is bound to the zeolite, i.e. the processes of desorption andadsorption of zeolite water are strictly reversible.

In touch with electrolyte solutions, cations from a zeolite (Breck, D.W., J. Chem. Educ., 41:678, 1964; Fedorov, V. A., Tolmachev, A. M.,Panchenkov, G. M., Zh. Fiz. Khim, 38:1248, 1964; Wolf, F., Foertig, H.,Kolloid Z.-Z. Polymere, 206:48, 1965, Sherry, H. S., Adv. Chem. Ser.,101:350, 1971, Brooke, N. M., Rees, L. V. C., Adv. Chem. Ser, 101:405,1971:Barrer, R. M., Klinowski, J., Phil. Trans 285:637, 1977). In theconditions of balance, the following applies: (Schwuger, M. J., Smolka,H. G, Tenside Detergents, 13:305, 1976):zB×A^(za)(aq)+zA×B^(zb)(s)⇄zB×A^(za)(s)+zA×B^(zb)(aq)where zA and zB are charge numbers (“valences”) of replaceable cations Aand B, while aq and s denote the solution, i.e., the firm phase(zeolite).

Natural zeolites are impurified with various admixtures, therefore weused a synthetic zeolite with strictly defined characteristics in thepreparation of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic presentation of mutual linking of and AlO₄tetrahedrons in the zeolite crystal grid.

FIG. 2 is a schematic presentation of cut cubo-octahedron (sodaliteunit) as a tertiary unit of the zeolite A structure (left) and thestructure of the unit cell of the zeolite A (right).

FIG. 3 is a schematic presentation of X and Y zeolites (faujasites) withthe bolonging S, sit, S_(r), S_(r1-) and SU positions of the Zeolite.

FIG. 4 depicts (a) 5-1 secondary unit of the mordenite structure, (b)projection of the cross-section of the crystal grid of mordenite alongthe axis of main channels, and (c) the schematic presentation of thecrystal grid of mordenite.

FIG. 5 depicts (a) the characteristic chain structure composed of 5-1secondary unit of the structure. (b) the crystal face (100) of a unitlattice of the ZSM-5 zeolite and silicalite. 10-member rings representopenings of sinusoidal channels parallel (001) with crystal faces of theZSM-S zeolite and silicalite, and (c) the schematic presentation ofstructural channels in the ZSM-5 zeolite (Si/Al—20-200) and silicalite(Si/Al—20-200) and the silicalite (SilAl=oo).

FIG. 6 is a chart showing the curve of the assimilation of glucose indiabetic CBA mice which were receiving the mineral part of MPP.

FIG. 7 is a chart showing the curve of the assimilation of glucose indiabetic NOD mice which were receiving the mineral pan of MPP

FIG. 8 is a chart showing the concentration of glucose in the blood ofcontrol diabetic mice (1) and diabetic mice treated with MPP (WorkingExample 16)

FIG. 9 is a chart showing the concentration of glucose in the blood ofdiabetic CBA mice non-treated with MPP (Control) and treated with MPP(Working Example 17).

FIG. 10 is a chart showing the quantity of water drunk during 6 days ofapplication of MPP (3 mice per group).

FIG. 11 is a chart showing the volume of excreted urine in diabetic micewhich were receiving MPP (3 mice per group) or 6 days.

FIG. 12 is a chart showing the concentration of fructosamine in theserum of control (Control), diabetic (Diab. Groups), and diabetic micetreated with MPP (Diab.T) according to Working Example 16.

FIG. 13 is a chart showing the catalytic concentration of GST in thegroup of healthy, control and diabetic CBA mice treated with MPP(Diab.+MPP) (According to the Working Example 16.

FIG. 14 depicts a cross-section through n. ischiadicus of diabetic miceafter 180 days of the duration of the disease.

FIG. 15 depicts a cross-section through n. ischiadicus of diabetic micetreated with MPP after 180 days of the duration of the disease.

DETAILED DESCRIPTION OF THE INVENTION Auxiliary Substances

In the production of pills and capsules, beside the MPP which is thesubject of this invention, usual auxiliary substances are used, whichare the carriers of certain functions:

Supplementing substances, e.g., preferably, lactose, microcrystallinecellulose, sorbitol, manitol, starch, etc.

Binding substances, e.g., preferably gelatin, cellulose,polyvinylpyrrolydon, methyl cellulose, etc.

Decomposition substances, e.g., preferably starch natrium-hydroxymethylstarch, microcrystalline cellulose, etc.

Sliding substances, e.g., preferably Mg-stearate, talc, stearine acid,hydrated plant oils.

The production of pills and capsules is carried out by the dryprocedure.

Characterization of Calcium Form of Synthetic Zeolite (Ca-zeolite (S))

The calcium form of the synthetic zeolite Ca-zeolite (S) which appearsin the form of fine white powder is characterized by methods of thex-ray diffraction, infrared spectroscopy, examination electronicmicroscopy, differential thermogravimetry and the distribution of thesize of particles.

WORKING EXAMPLE 1 X-ray Diffraction Analysis

X-ray diffractograms of the Ca-zeolite (S) were obtained by the Philips'diffractometer with CuKα radiation within the field of Bragg angles20=10°-46° have shown that all the analyzed samples were 95–100%crystalline and without admixtures of other non-zeolite crystallinephases.

WORKING EXAMPLE 2 Infrared Spectroscopic Analysis

Infrared spectres of the Ca-zeolite (S) were recorded by the techniqueof the KBr pastille on the Perkin-Elmer infrared spectrometer System2000 FT-IR, which showed that all the samples had spectrescharacteristic for zeolites, without admixtures of other non-zeolitecrystalline phases.

WORKING EXAMPLE 3 Simultaneous Thermic Analysis

The simultaneous thermic analysis of the Ca-zeolite (S) was performed bythe device TA 4000 System (Mettler-toledo), the speed of warming up inthe atmosphere of nitrogen was 10 K/min. The results of the analysishave shown that the analyzed samples contain 12.5–22.6% of H₂O.

WORKING EXAMPLE 4 Measuring of Particle Size

Distributions of the sizes of particles of the Ca-zeolite (S) weremeasured by the method of dynamic scattering of the laser light by thedevice Mastersize X (Malvern). The results of the analyses have shownthat the size of particles of the analyzed samples amounts to 0.5–20micrometers.

WORKING EXAMPLE 5 Chemical Analysis

The chemical analysis of the Ca-zeolite (S) was carried out in thefollowing way: Certain quantities of the zeolite were dissolved in thediluted solution of nitric acid. The solutions obtained in that way werediluted with distilled water up to levels suitable for measuring ofsodium, aluminum and silicium concentrations by the method of atomicabsorption spectroscopy (AAS). The acid stable zeolites were melted withthe mixture of sodium carbonate and sodium tetraborate. The melt wasdissolved in the diluted solution of HCl and diluted with distilledwater up to the level suitable for measuring of the sodium, aluminum andsilicium concentrations by the AAS method.

The concentrations of sodium, aluminum and silicium in the statedsolutions were measured by the atomic absorption spectrophotometer 3030B(Perkin-Elmer). The results of the chemical analysis have showed thatthe analyzed samples contain 6.5–15.6% of CaO, 11.8–28.4% of Al₂O₃,33.5–69.3% SiO₂.

Preparation of Oligopeptides

WORKING EXAMPLE 6 Isolation of Proteins of Nettle Root, Stalk and Leaf

Urticae Radix, Herba Et Flos

Urtica Dioica L., Urticaceae-Nettle-Nettle

From 1 kg of root, stalk and leaf, cut into tiny pieces, an alcoholicextraction was made through 5 days. By cold distillation, alcohol wasremoved from the extraction. By further procedure, oligopeptides ofnettle were froze and lyophilized. In order to remove impuritiescontained by the plant extract, lyophilized nettle extract was appliedon the column Sephadex G-25. Protein components were eluted from thecolumn by the use of Tris-HCl buffer as the fraction 12. The absorptionof protein was measured at 280 nm. Collected fractions from 1 ml eachwere submitted to the dialysis and lyophilized. The qualitative systemof amino acids was determined on a thin layer of cellulose (Merck), inthe mixture of the solvent: n-butanol:acetone acetic acid:water,

WORKING EXAMPLE 7 Isolation of Proteins of Milk Vetch Root

Astragali Radix

Astragalus Membranaceus (Fisch. Ex Link) Fabaceae—Milk Vetch

From 1 kg of the root of milk vetch, cut to tiny pieces, an alcoholicextraction was made through 5 days. By cold distillation, alcohol wasremoved from the action. By further procedure, olygopeptides of milkvetch were frozen and lyophilized. In order to remove impuritiescontained by the plant extract, lyophilized milk vetch extract wasapplied on the column Sephadex G-25. Protein components were eluted fromthe column by the use of Tris-HCl buffer as the fraction 11. Theabsorption of protein was measured at 280 nm. Collected fractions from 1ml each were submitted to the dialysis and lyophilized. The qualitativesystem of amino acids was determined on a thin layer of cellulose(Merck), in the mixture of the solvent: n-butanol:acetone aceticacid:water.

WORKING EXAMPLE 8 Isolation of Proteins of Balm

Melissa Officinalis L.—Balm

From 1 kg of the stalk and leaves of balm, cut to tiny pieces, analcoholic extraction was made through 5 days. By cold distillation,alcohol was removed from the extraction. By further procedure,olygopeptides of balm were frozen and lyophilized. In order to removeimpurities contained by the plant extract, lyophilized balm extract wasapplied on the column Sephadex G-25. Protein components were eluted fromthe column by the use of Tris-HCl buffer as the fraction 10. Theabsorption of protein was measured at 280 nm. Collected fractions from 1ml each were submitted to the dialysis and lyophilized. The qualitativesystem of amino acids was determined on a thin layer of cellulose(Merck), in the mixture of the solvent: n-butanol:acetone aceticacid:water.

WORKING EXAMPLE 9 Isolation of Proteins of Hop

Humulus Lupulus L.—Hop

From 1 kg of the cones of hop, cut to tiny pieces, an alcoholicextraction was made through 5 days. By cold distillation, alcohol wasremoved from the extraction. By further procedure, olygopeptides of hopwere frozen and lyophilized. In order to remove impurities contained bythe plant extract, lyophilized hop extract was applied on the columnSephadex G-25. Protein components were eluted from the column by the useof Tris-HCl buffer as the fraction 12. The absorption of protein wasmeasured at 280 nm. Collected fractions from 1 ml each were submitted tothe dialysis and lyophilized. The qualitative system of amino acids wasdetermined on a thin layer of cellulose (Merck), in the mixture of thesolvent: n-butanol:acetone acetic acid:water.

Preparation of Vitamins

WORKING EXAMPLE 10 Preparation of B Complex

As the basic source of vitamins of the B complex was used an inactivepreparation of yeasts rich in proteins, hydrocarbons, lipids, minerals,vitamins and essential amino acids. This preparation was used as thebasic source of the B complex vitamins in the MPP.

WORKING EXAMPLE 11 Preparation of MPP

The MPP is prepared by mixing of the calcium form of the artificialzeolite, proteins of: nettle, milk vetch, balm and hop and the B-complexvitamins.

The MPP prepared in that way contains: 10 to 50% (m/m) of the calciumform of the zeolite.

(CaO.Al₂O₃.ySiO₂.z′H₂O), 0.5 to 20% (m/m) of the protein of the milkvetch root, 0.5 to 20% (m/m) of the protein of balm, 0.5 to 20% (m/m) ofthe protein of hop, 0.5 to 17.5% (m/m) of the protein of nettle and 0.5to 12.5% (m/m) of B-complex.

WORKING EXAMPLE 12 Experimental Diabetes

Tests were made on two models of experimental diabetes.

Experimental diabetes was caused by alloxan in CBA mice, in the dose of75 mg/kg of body weight. After the appearance of the symptoms ofdiabetes, 3 mice were kept in each cage.

NOD mice, which developed all the symptoms of diabetes, were taken inthe experiment.

In pharmalogical tests, the mineral-herbal preparation was admixed tothe standard food for laboratory mice.

This invention will now be shown with particular examples showing that,in case of diabetes, a syndrome is in question, and that for asuccessful treatment of Type I or II Diabetes, it is not sufficient toapply the known medicine which has only the characteristic of a stronghypoglycemic effectiveness, but that the mineral-herbal preparation fromthe invention should be applied which helps the disturbed metabolism inits entirety.

WORKING EXAMPLE 13 Determining of the Level of Apoptosis

The level of apoptosis was determined after cutting of the sample of thenerve in cryostat. On the cut samples of 4 μm, propidium iodine wasadded and the sample was analyzed under a fluorescent microscope.

WORKING EXAMPLE 14 Presence of IgG on Nerves

The presence of IgG on nerves was ascertained by colouring of nerveswith peroxidase antiperoxidase and the analyzed under a microscope.

WORKING EXAMPLE 15

Diabetic animals which were receiving the MPP with the composition ofminerals from 0.5 to 10 mg, proteins of milk vetch from 0.1 to 2 mg,proteins of nettle 0.1 do 1.5 mg, proteins of balm 0.1 to 2.0 mg,proteins of hop 0.1 to 2.0 mg, B1 vitamin from 0.5 to 10 μg, B2 from 2to 10 μg, B6 from 0.5 to 1.25 μg, B12 from 0.3 to 1 μg, with all thesymptoms of diabetes, with over 19.0 mmol/L of glucose in blood. Micewere placed in metabolic cages and, during 6 days, the quantity of waterdrunk, the quantity of food eaten, of urine and feces excreted weremeasured. During the first three days of the application of the MPP, theanimals did not show a reduction of the symptoms of diabetes. The samewas repeated in the next three days. The concentration of glucose inblood was above 16 mmol/L, and the animals drank over 25 ml of waterdaily. Further therapy had a positive effect on symptoms of diabetes inCBA and NOD mice. During the test, the mice were moderately active.

WORKING EXAMPLE 16

Diabetic animals which were receiving the MPP with the composition ofminerals from 10 to 50 mg, proteins of milk vetch from 1 to 10 mg,proteins of nettle from 1 do 12 mg, proteins of balm 0 to 10 mg,proteins of hop 0 to 10 mg, B1 vitamin of 25 μg, B2 of 18 μg, B6 of 2.5μg, B12 of 0.7 μg, with all the symptoms of diabetes, with over 14.5mmol/L of glucose in blood. Mice were placed in metabolic cages and,during 6 days, the quantity of water drunk, the quantity of food eaten,of urine and feces excreted were measured. During the first three daysof the application of the MPP, the animals considerably reduced thesymptoms of diabetes. The same was repeated in the next three days, sothat the volume of water drank and urine excreted was reduced by 60%. Inthe test of the effectiveness of the MPP on the concentration of glucosein the peripheral blood of diabetic mice, the preparation showed ahypoglycemic effect. Besides, the curve of the assimilation of glucosewas considerably more favourable in relation to untreated controlgroups, which is valid both for CBA diabetic mice, and for NOD diabeticmice. During the further therapy, through 6 months, symptoms of diabeteswere not completely removed, but they were considerably reduced. Itshould be particularly pointed out here that, beside the reduction ofthe concentration of glucose in blood, a considerable reduction of latecomplications of diabetes, e.g.: neuropathies, arose. During the tests,the mice were moderately active.

WORKING EXAMPLE 17

Diabetic animals which were receiving the MPP with the composition ofminerals from 50 to 500 mg, proteins of milk vetch from 10 to 100 mg,proteins of nettle from 10 do 87.5 mg, proteins of balm 0 to 100 mg,proteins of hop 0 to 100 mg, B1 vitamin from 0.5 to 10 μg, B2 from 2 to10 μg, B6 from 0.5 to 1.25 μg, B12 from 0.3 to 1 μg, with all thesymptoms of diabetes, with over 16 mmol/L of glucose in blood. Mice wereplaced in metabolic cages and, during 6 days, the quantity of waterdrunk, the quantity of food eaten, of urine and feces excreted weremeasured. During the first three days of the application of the MPP, theanimals considerably reduced the symptoms of diabetes. The same wasrepeated in the next three days, so that the volume of water drank andurine excreted was reduced by 70%. In the test of the effectiveness ofthe MPP on the concentration of glucose in the peripheral blood ofdiabetic mice, the MPP showed a hypoglycemic effect. However, the testof burdening with glucose did not show a positive effect of thepreparation and the curve of the assimilation of glucose was notconsiderably more favourable in relation to unrelated control groups,which is valid both for CBA diabetic mice, and for NOD diabetic mice.During the further therapy, through 6 months, symptoms of diabetes werenot completely removed, but they were considerably reduced. It should beparticularly pointed out here that, for the purpose of reduction of thelevel of glucose, a considerable moves occurred, besides, the symptomsof diabetes were considerably reduced. During the test, the mice weremoderately active.

Pharmacological Data

The effectiveness of the MPP on the concentration of glucose in bloodwas tested on control and diabetic CBA and NOD mice. The results werecompared with the control group of diabetic CBA mice which were notreceiving the MPP.

The animals were receiving the MPP by a probe during 14 days. On the14^(th) day, the zero blood sample (25 μl.) was taken from the tailvein. Firstly, the mice were submitted to testing of the effectivenessof the MPP, the mineral preparation (Working Example 1 to 3), secondly,of the effect of the mineral preparation plus plant proteins (WorkingExample 4 to 15).

The results of burdening with glucose were presented in FIGS. 6 and 7.

The concentration of glucose in the blood of diabetic CBA mice (10 miceper group) which were not receiving the mineral part of the MPPaveragely amounted to 19.8±3.2 mmol/L (FIG. 6). After 10, i.e. 30minutes from the initiation of glucose (1 g/kg of body weight), theconcentration of glucose in the peripheral blood of diabetic mice wasstrongly increased in value above 30 mmol/L. During the next half anhour, the concentration of glucose stated to be decreased, but after anhour's time, it did not reach the beginning values of the concentrationof glucose in blood (FIG. 6).

In the group of diabetic mice which were receiving the mineralpreparation MPP, the concentration of glucose in blood after initiatingof glucose per os was strongly increased from 18 mmol/L to 27 mmol/Lafter 10 minutes. During the next 50 minutes, the concentration ofglucose in the peripheral blood of diabetic mice was continuouslydecreasing (FIG. 6).

In the group of diabetic NOD mice, the test of burdening with glucoseshowed that the concentration of glucose in blood returned to thebeginning values after 60 min. It can be concluded that the diabetic NODmice which were receiving the mineral part of the MPP by probeassimilate the introduced glucose much better (FIG. 7). The mineral MPPpreparation did not show a direct hypoglycemic effect.

But, if diabetic mice were receiving MPP for 14 days, the concentrationof glucose in the blood of those mice is presented in FIG. 8. Beforeprobing with MPP, a sample of blood was taken and the zero value of theconcentration of glucose was determined. During the next 4 hours ofprobing, in certain periods of time, a sample of their blood was taken,the concentration of glucose in diabetic mice was continuously fallingand, after 4 hours, it amounted to only about 20% of the beginning value(FIG. 8). The mineral protein preparation according to the WorkingExample 16 showed a strong hypoglycemic effect during 4 hours oftesting.

In diabetic mice which were receiving the MPP during 14 days (accordingto Working Example 17), the concentration of glucose in blood duringfour hours after introducing of the MPP was not changing in relation tocontrol values. (FIG. 9).

Diabetic CBA mice which were receiving MPP during 6 days considerablyreduced the volume of the drunk water (p>0.001) (FIG. 10).

Diabetic CBA mice which were receiving MPP during 6 days considerablyreduced the volume of the secreted urine (p>0.001) (FIG. 11).

During 6 months, mice were daily together with the MPP food. From Table1, it is evident that the MPP has not shown a harmful effect because thebody weight of the controlled, healthy mice was not being decreased, butit was increased during the experiment. Thus, the body weight wasaveragely increased from the beginning 25 grams to 39 grams.

But in the group of diabetic CBA mice, there was no increase of the bodyweights and it was averagely around 26 grams (Table 1). During the wholeexperiment, there was no increase of the body weight from the beginningvalues (Table 1), which is a usual occurrence in diabetes.

TABLE 1 The body weights of control and diabetic CBA mice which werereceiving the MPP. Months Groups 0 1 2 3 4 5 6 Control 24.3 ± 1.3 26.9 ±2.1 29.7 ± 2.3 32.5 ± 2.8 33.6 ± 2.5 36.3 ± 2.2 39.3 ± 2.8 Diabetic CBA27.7 ± 2.2 26.1 ± 1.2 26.2 ± 3.2 25.9 ± 3.7 25.7 ± 3.3 26.6 ± 2.8 26.5 ±2.2

During the experiment, a decrease of the body weight was noticed in thegroup of diabetic NOD mice (Table 2).

In the beginning of the experiment, the body weight was about 35 grams,while mice were averagely weighing 30 grams (Table 2).

In the group of control, non-diabetic NOD mice, the body weight duringthe experiment was continuously increasing (Table 2).

The concentration of fructosamine in the serum of diabetic NOD mice wasdetermined for the purpose of monitoring the efficiency of the treatmentof diabetes with the MPP. The results showed that the concentration offructosamine in the control group of NOD mice amounts to 219 μmol/L. Inthe serum of diabetic NOD mice, the concentration of fructosamine issignificantly increased. FIG. 12 (p<0.001). In the group of diabetic NODmice treated with MPP, the measured concentration of fructosamineamounted to averagely 240 μmol/L, which is considerably lower inrelation to the diabetic group (p<0.001) (FIG. 12).

In the cytosol of the liver of experimental groups, the effect of themineral-plant preparation on the catalytic concentration of glutationS-transferase (GST) was determined.

GST is an enzyme of Phase II of metabolizing of xenobiotics and itparticipates in their detoxication and thus protects the station fromthe toxic effect of electrophilic substances. It catalyzes the reactionof conjugation of glutations with a large number of electrophiliccomponents arisen in the cell.

The catalytic concentration of GST in the group of healthy, control anddiabetic CBA mice amounted averagely to 17.19 nmol/min/mg (FIG. 13). Inthe group of healthy CBA mice treated with MPP, the catalyticconcentration of GST amounted averagely to 16.42 nmol/min/mg. Aconsiderable increase of the catalytic concentration of GST amountedaveragely to 16.42 nmol/min/mg. A considerable increase of the catalyticconcentration of GST arose in the group of diabetic CBA mice (p>0.05).After the six-month treatment of diabetic CBA mice with MPP, aconsiderable decrease of the catalytic concentration of GST in the liveroccurred to averagely 12.23 nmol/min/mg, which is at the level ofsignificance of p>0.01 a considerable difference in comparison with thediabetic group (FIG. 13).

During 6 months of testing of the effect of the MPP on the diabeticcondition, the level of the concentration of calcium ions (Ca²⁺) in theserum of control and diabetic mice was monitored (Table 3).

The concentration of (Ca²⁺) ions during 6 months was not changed in theblood of diabetic CBA mice which were receiving the MPP daily (accordingto the Working Example 16). But in the group or diabetic CBA mice, asignificant decrease in the concentrations of (Ca²⁺) ions in the serumarose (p>0.05) after 6 months of the duration of the disease. Similarresults were shown by mice with the spontaneous diabetes (Table 4). Theconcentration of (Ca²⁺) ions was not changed in the group of diabeticNOD mice which were receiving the MPP daily. A considerable reduction ofthe concentration of calcium ions was measured in the group of diabeticNOD mice which were not receiving the MPP (p>0.005) (Table 4).

TABLE 2 The body weights of control and diabetic NOD mice which werereceiving the MPP. Months Groups 0 1 2 3 4 5 6 NOD 34.3 ± 3.8 33.7 ± 3.331.1 ± 4.1 30.5 ± 4.1 31.2 ± 4.4 30.9 ± 3.6 30.5 ± 3.2 NOD 30.4 ± 2.632.8 ± 3.4 35.6 ± 1.0 36.5 ± 2.3 37.8 ± 2.3 38.1 ± 2.6 39.4 ± 3.4non-diabetic

TABLE 3 Concentration of Ca²⁺ in the serum of control and diabetic CBAmice which were receiving the MPP during 180 days. Days Groups 7 90 180Diabetes  2.2 ± 0.05  2.0 ± 0.06 1.92 ± 0.12* Diabetes + MPP 2.17 ± 0.032.19 ± 0.05 2.23 ± 0.2 

TABLE 4 Concentration of Ca²⁺ in the serum of control and diabetic CBAmice which were receiving the MPP during 180 days. Days Groups 7 90 180NOD 2.2 ± 0.08 2.01 ± 0.08 1.90 ± 0.04* NOD + MPP 2.2 ± 0.09 2.15 ± 0.032.16 ± 0.07 

Animals form particular groups were sacrificed after 7, 90, that is 180days. By patho-histological treatment, the level of neuropathy of thedigestive tract (Tables 5 and 6) was ascertained. After 180 days,diabetic CBA mice which were not receiving the MPP, developed diabeticneuropathy in 100% of cases. However, in diabetic CBA mice which werereceiving the MPP each day, the diabetic neuropathy was not observed(Table 5).

TABLE 5 Presence of neuropathy of the gastro-intestinal tract indiabetic CBA mice during 180 days of the duration of the experiment.Days Groups 7 90 180 Diabetes − + +++ Diabetes + MPP − + +

The same experiment was also done with diabetic NOD mice. Diabetic NODmice develop diabetic neuropathy in 100% of cases during 6 months (Table6). Diabetic NOD mice, which were receiving the MPP during 180 days eachday did not develop diabetic neuropathy (Table 7).

TABLE 6 Presence of neuropathy of the gastro-intestinal tract indiabetic NOD mice during 180 days of the duration of the experiment.Days Groups 7 90 180 NOD + ++ +++ NOD + MPP + + +

In order to check the extent of the damage of nerves, i.e. to examinethe presence of apoptotic bodies, on the day of sacrificing, samples ofn. ischiadicus were taken from mice. The obtained results show thatlong-term diabetes has a considerable effect on the increase of thenumber of apoptotic bodies. In the group of diabetic mice, the number ofapoptotic bodies was about 67% (Table 7). In controls (healthy mice),the number of apoptotic bodies was about 9%. Diabetic mice treated withthe MPP did not show a considerable increase of the number of apoptoticbodies in n. ischiadicus (16%)(Table 7).

By peroxidase antiperoxidase colouring, the presence of antibodies (IgG)on nervus ischiadicus was tested. Diabetic mice which developed all thesymptoms of diabetic neuropathy during the experiment showed a strongpresence of IgG in n. ischiadicus (FIG. 14). Contrary to that, micewhich were receiving the MPP for 6 months did not have auto-antibodieson nerve fibres of n. ischiadicus (FIG. 15).

The percentage of apoptotic bodies is statistically considerably smallerin the group of mice (CBA and NOD) treated by the MPP (Tables 7 and 8).Further, after colouring with anti IgG conjugated with peroxidaseantiperoxidase, the percentage of positive neurons in the group of micetreated by the MPP was between 6 and 9% (Tables 7 and 8).

TABLE 7 Percentage of apoptotic bodies, and the percentage of IgGpositive nerve fibres in CBA mice after 6 months (180 days) of testing.Days Groups Apoptosis (%) IgG (%) Diabetes 67 86 Diabetes + MPP 16  9

TABLE 8 Percentage of apoptotic bodies, and the percentage of IgGpositive nerve fibres in NOD mice after 6 months (180 days) of testing.Days Groups Apoptosis (%) IgG (%) NOD 78 91 NOD + MPP 12  6 No harmfultoxic effects were ascertained if the animals were receiving the MPPduring 6 months.

CONCLUSION

Since neuropathy is a late complication often present in diabetes, itwas necessary to find the additional “means” to the therapy which willmake the overall state of the patient considerably easier. For thatpurpose, the technical problem which was originally set before theinventor and finally successfully resolved resulted in the currentinvention of the mineral-protein preparation against neuropathies indiabetes with the following characteristics:

-   1) it reduces the concentration of glucose by its absorption on the    MPP, which is evidenced by the figures showing the assimilation of    glucose,-   2) it stops the process of depositing of Ca²⁺ ions into nerve cells,-   3) it reduces the process of apoptosis in nerve cells stimulated by    depositing of Ca²⁺ ions,-   4) the process of phagocytosis of apoptotic bodies is absent,-   5) it stops the process of creating immunoglobulin on myelin fibres    of the nerve membrane,-   6) it reduces the depositing of immunoglobulin on nerve fibres,-   7) it reduces the immunological destruction of nerve fibres mediated    by the complement,-   8) the pain caused by diabetic neuropathy is absent,-   9) it contains the necessary active substances for elimination of    neurotic digestive troubles,-   10) it contains the necessary active substances for elimination of    neurotic heart troubles,-   11) it contains the necessary active substances for elimination of    harmful radicals,-   12) it contains the necessary B complex vitamins of natural origin,-   13) mineral particles do not pass through villi,-   14) mineral particles do not pass through the blood vessel barrier,-   15) mineral particles do not pass through the intestine blood    barrier,-   16) the nature of the material is a strong ionic exchange,-   17) it is suitable for oral administration and it had no harmful    side effects during testing,-   18) no noticeable harmful side effects appear even with large daily    doses, and in case of a long-term use,-   19) it is not harmful in case of its long-term use.

Working examples are given only as an illustration and they do notrepresent a limitation of the MPP, the range of which is determined bythe contents of patent applications.

1. A mineral-protein preparation, comprising synthetic Ca-zeolite,protein extract of nettle, protein extract of milk vetch, proteinextract of balm, protein extract of hop cones, and a source of B-complexvitamins.
 2. The mineral-protein preparation according to claim 1comprising synthetic Ca-zeolite 10–50 m/m % protein extract of nettle0.5–17.5 m/m % protein extract of milk vetch 0.5–20 m/m % proteinextract of balm 0.5–20 m/m % protein extract of hop cones 0.5–20 m/m % asource of B-complex vitamins 0.5–12.5 m/m %.


3. The mineral-protein preparation according to claim 2 comprisingsynthetic Ca-zeolite 40 m/m % protein extract of nettle 15 m/m % proteinextract of milk vetch 15 m/m % protein extract of balm 10 m/m % proteinextract of hop cones 10 m/m % a source of B-complex vitamins 10 m/m %.


4. The mineral-protein preparation of claim 2 wherein the syntheticzeolite was prepared by crystallization of an aluminosilicate hydrogelobtained by mixing an alkaline solution of sodium aluminate and analkaline solution of sodium silicate after which ionic exchange of thesynthetic zeolite was carried out, during which Na⁺ ions were replacedwith Ca²⁺ ions.
 5. The mineral-protein preparation of claim 4, whereinthe sodium content of the synthetic zeolite is reduced by procedures ofionic exchange to 7.2% from 17.2% of Na⁺ ions, calculated as Na₂O andthe calcium content is increased to 15.6% from 6.5% Ca²⁺ ions,calculated as CaO.
 6. The mineral-protein preparation according to claim5, comprising  6.5–15.6% CaO, 11.8–28.4% Al₂SO₃, 33.5–69.3% SiO₂, and12.5–22.6% H₂O.


7. The mineral-protein preparation according to claim 6 wherein thecalcium synthetic zeolite is 95–100% crystalline and without admixturesof other non-zeolite crystalline phases.
 8. The mineral-proteinpreparation according to claim 6, wherein the synthetic zeolite ispresent in particles ranging in size from 0.5 to 20 micrometers.
 9. Themineral-protein preparation according to claim 8, wherein the specificsurface of the synthetic zeolite particles is from 0.15 to 6 m²/g. 10.The mineral-protein preparation according to claim 2 wherein the proteinextracts are obtained by the purification of the alcoholic extract ofeach on Sphadex G-25.
 11. The mineral-protein preparation according toclaim 2 wherein the source of the B-complex vitamins contained in thepreparation is inactive yeast.
 12. The mineral-protein preparationaccording to claim 2 wherein the inactive yeast is Saccharomices Sp. 13.A medicament comprising the preparation of claim 1 and physiologicallyacceptable auxiliary substances.
 14. The medicament of claim 13formulated for peroral administration.
 15. The medicament of claim 14 intablet form.
 16. A method of treating a diabetic mammal to reduce one ormore of the concentration of GUK, fluid intake, the process of apoptosisin nerve cells, the process of creating of IgG on myelin fibres of nervemembranes or the process of phagocytosis of apoptotic bodies comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the medicament of claim
 13. 17. A method for the treatment ofdiabetic neuropathies comprising administering to a patient in needthereof a therapeutically effective amount of the medicament of claim13.
 18. A method of reducing, in a diabetic mammal, one or more of theconcentration of glucose in the blood, the amount of fructosamine in theblood, the concentration of GST in the liver or minimizing in a diabeticmammal one or more of the presence of apoprotic bodies or diabeticneuropathy of the digestive tract, or maintaining the level of Ca²⁺ ionsin the serum of a diabetic mammal, comprising administering to a patientin need thereof a therapeutically effective amount of the medicament ofclaim 13.